In mammals, the Sirtuins are composed of seven Sir2 orthologues (Sirt1-7) with a conserved deacetylase core that utilizes NAD+ as a cofactor. Sirtuins play an important role in energy metabolism and regulating Sirtuin activity may be useful for controlling fat accumulation. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 a.a. sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an on switch for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.